Dna sequencing by nanopore using modified nucleotides

ABSTRACT

This invention provides a process for sequencing single-stranded DNA or RNA by employing a nanopore and modified nucleotides.

This application is a continuation of U.S. application Ser. No.15/255,029, filed Sep. 1, 2016, which is a continuation of U.S.application Ser. No. 14/516,785, filed Oct. 17, 2014, which is acontinuation of U.S. application Ser. No. 12/308,091, filed Dec. 4,2008, now U.S. Pat. No. 8,889,348, issued on Nov. 18, 2014, which is a §371 national stage of PCT International Application No.PCT/US2007/013559, filed Jun. 7, 2007, and claims the benefit of U.S.Provisional Application No. 60/811,912, filed Jun. 7, 2006, the contentsof all of which are hereby incorporated by reference into thisapplication.

This invention was made with government support under grant HG003718awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named “181212 76315-AAAA-PCT-USSubstitute Sequence Listing RBR.txt”, which is 3 kilobytes in size, andwhich was created Dec. 12, 2018 in the IBM-PC machine format, having anoperating system compatibility with MS-Windows, which is contained inthe text file that was filed Dec. 12, 2018 as part of this application.

Throughout this application, various publications are referenced inparentheses by number. Full citations for these references may be foundat the end of the specification immediately preceding the claims. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

DNA sequencing is a fundamental technology for biology. Severalanalytical methods have been developed to detect DNA or RNA at thesingle molecule level using chemical or physical microscopictechnologies [15, 16, 21 and 23]. In the past few years, the ion channelhas been explored for detecting individual DNA or RNA strands, withnanopore being a candidate for high rate sequencing and analysis of DNA[9, 10, 4, 3 and 7].

In 1996, Kasianowicz et al. first demonstrated that the α-hemolysinchannel, an exotoxin secreted by a bacterium, could be used to detectnucleic acids at the single molecule level [ 8]. The monomericpolypeptide self-assembles in a lipid bilayer membrane to form aheptameric pore, with a 2.6 nm-diameter vestibule and 1.5 nm-diameterlimiting aperture (namely, the narrowest point of the pore) [1, 14 and15]. In an aqueous ionic salt solution such as KCl, the pore formed bythe α-hemolysin channel conducts a sufficiently strong and steady ioniccurrent when an appropriate voltage is applied across the membrane. Thelimiting aperture of the nanopore allows linear single-stranded but notdouble-stranded nucleic acid molecules (diameter −2.0 nm) to passthrough. The polyanionic nucleic acids are driven through the pore bythe applied electric field, which blocks or reduces the ionic currentthat would be otherwise unimpeded. This process of passage generates anelectronic signature (FIG. 1) [23 and 5]. A particular nucleic acidmolecule, when entering and passing through the nanopore, will generatea characteristic signature that distinguishes it from others. Theduration of the blockade is proportional to the length of the nucleicacid, and its signal strength is related to the steric and electronicproperties of the nucleotides, namely the identity of the four bases (A,C, G and T).

A specific event diagram is constructed which is the plot oftranslocation time versus blockade current. This specific event diagram(also referred to as an electronic signature) is used to distinguish thelengths and the compositions of polynucleotides by single-channelrecording techniques based on characteristic parameters such astranslocation current, translocation duration, and their correspondingdispersions in the diagram [14].

Although the nanopore approach is known as a DNA detection method, thisapproach for base-to-base sequencing has not yet been achieved.

SUMMARY OF THE INVENTION

This invention provides a method for determining the nucleotide sequenceof a single-stranded DNA comprising the steps of:

-   -   (a) passing the single-stranded DNA through a pore of suitable        diameter by applying an electric field to the DNA, wherein at        least each A or each G residue and at least each C, each T or        each U residue comprises a modifying group bound to its        respective base so that each type of nucleotide in the DNA has        an electronic signature which is distinguishable from the        electronic signature of each other type of nucleotide in the        DNA;    -   (b) for each nucleotide of the DNA which passes through the        pore, determining an electronic signature for such nucleotide;        and    -   (c) comparing each electronic signature determined in step (b)        with electronic signatures corresponding to each of A, G, C and        T modified as per the nucleotides in the single-stranded DNA, so        as to determine the identity of each such nucleotide,        thereby determining the nucleotide sequence of the        single-stranded DNA.

This invention also provides a method for determining the nucleotidesequence of a single-stranded RNA comprising the steps of:

-   -   (a) passing the single-stranded RNA through a pore of suitable        diameter by applying an electric field to the RNA, wherein at        least each A or each G residue and at least each C or each U        residue comprises a modifying group bound to its respective base        so that each type of nucleotide in the RNA has an electronic        signature which is distinguishable from the electronic signature        of each other type of nucleotide in the RNA;    -   (b) for each nucleotide of the RNA which passes through the        pore, determining an electronic signature for such nucleotide;        and    -   (c) comparing each electronic signature determined in step (b)        with electronic signatures corresponding to each of A, G, C and        U modified as per the nucleotides in the single-stranded RNA, so        as to determine the identity of each such nucleotide,        thereby determining the nucleotide sequence of the        single-stranded RNA.

This invention also provides a nucleotide having an azido groupcovalently bound to its base.

This invention also provides a method for making a modified nucleotidecomprising contacting the instant nucleotide with an alkyne-containingcompound under conditions permitting reaction between the azido and thealkyne groups, thereby making the modified nucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. α-Hemolysin protein self-assembles in a lipid bilayer to form anion channel and a nucleic acid stretch passes through it (left), withthe corresponding electronic signatures generated (right) [23 and 5].

FIG. 2. Structures of nucleotides dATP, dGTP, dCTP and dTTP.

FIG. 3. Nucleotide bulkiness in ascending order: (a) 5′-C₆₀T-3′, (b)5′-G*₆₀T-3′, (c) 5′-A*₆₀T-3′, and (d) 5′-T*₆₀T-3′.

FIG. 4. Structures of dCTP and dGTP, and modified nucleotides (dATP-NH₂and dUTP-NH₂).

FIG. 5. Modification of dATP-NH₂ and dUTP-NH₂.

FIG. 6. DNA-extension reaction using modified nucleotides (dATP-NHCOR₁and dUTP-NHCOR₂) to generate a modified single-stranded DNA chain. (SEQID NOs. 1 and 2 for template and primer, respectively).

FIG. 7. Steps of verifying sequencing capacity via nanopore usingvarious DNA sequences. (SEQ ID NOs. 3-6 for part (i), top to bottom,respectively; SEQ ID NO:7 for part (ii); SEQ ID NO:8 for part (iii); andSEQ ID NO:9 for part (iv)).

FIG. 8. Structures of unmodified nucleotides (dCTP and dGTP) andhook-labeled nucleotides (dATP-NH₂ and dUTP-N₃). The amino and the azidogroups function as hooks to conjugate with bulky groups after thenucleotides are incorporated into the DNA strand.

FIG. 9. Synthesis of dUTP-N₃.

FIG. 10. DNA-extension reaction using hook-labeled nucleotides (dATP-NH₂and dUTP-N₃) to generate a modified single-stranded DNA chain, whichwill then react with large functional groups (R1 and R3) selectively fordistinct detection by nanopore. (SEQ ID NOs. 1 and 2 for template andprimer, respectively)

DETAILED DESCRIPTION OF THE INVENTION Terms

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

A—Adenine; C—Cytosine;

DNA—Deoxyribonucleic acid;

G—Guanine;

RNA—Ribonucleic acid;

T—Thymine; and U—Uracil.

“Electronic signature” of a nucleotide passing through a pore viaapplication of an electronic field shall include, for example, theduration of the nucleotide's passage through the pore together with theobserved amplitude of current during that passage. Electronic signaturescan be visualized, for example, by a plot of current (e.g. pA) versustime. Electronic signature for a DNA is also envisioned and can be, forexample, a plot of current (e.g. pA) versus time for the DNA to passthrough the pore via application of an electric field.

“Nanopore” includes, for example, a structure comprising (a) a first anda second compartment separated by a physical barrier, which barrier hasat least one pore with a diameter, for example, of from about 1 to 10nm, and (b) a means for applying an electric field across the barrier sothat a charged molecule such as DNA can pass from the first compartmentthrough the pore to the second compartment. The nanopore ideally furthercomprises a means for measuring the electronic signature of a moleculepassing through its barrier. The nanopore barrier may be synthetic ornaturally occurring in part. Barriers can include, for example, lipidbilayers having therein α-hemolysin, oligomeric protein channels such asporins, and synthetic peptides and the like. Barriers can also includeinorganic plates having one or more holes of a suitable size. Herein“nanopore”, “nanopore barrier” and the “pore” in the nanopore barrierare sometimes used equivalently.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

“Type” of nucleotide refers to A, G, C, T or U.

Embodiments of the Invention

This invention provides a method for determining the nucleotide sequenceof a single-stranded DNA comprising the steps of:

-   -   (a) passing the single-stranded DNA through a pore of suitable        diameter by applying an electric field to the DNA, wherein at        least each A or each G residue and at least each C, each T or        each U residue comprises a modifying group bound to its        respective base so that each type of nucleotide in the DNA has        an electronic signature which is distinguishable from the        electronic signature of each other type of nucleotide in the        DNA;    -   (b) for each nucleotide of the DNA which passes through the        pore, determining an electronic signature for such nucleotide;        and    -   (c) comparing each electronic signature determined in step (b)        with electronic signatures corresponding to each of A, G, C and        T modified as per the nucleotides in the single-stranded DNA, so        as to determine the identity of each such nucleotide,        thereby determining the nucleotide sequence of the        single-stranded DNA.

In an embodiment of the instant method, the single-stranded DNA isobtained by (a) synthesizing double-stranded DNA using a single-strandedtemplate, a DNA polymerase and nucleotides, wherein at least each A oreach G residue and at least each C or each T residue comprises amodifying group bound to its respective base so that each type ofnucleotide in the DNA has an electronic signature which isdistinguishable from the electronic signature of each other typenucleotide in the DNA, and (b) removing from the resultingdouble-stranded DNA the single-stranded DNA containing modifiednucleotides.

In another embodiment of the instant method, the single-stranded DNA isobtained by (a) synthesizing double-stranded DNA using a single-strandedtemplate, a DNA polymerase and nucleotides, wherein at least each A,each G, each C, each U or each T residue comprises an azido group boundto its base, and at least each A, each G, each C, each U and each Tcomprises an amino group bound to its base, whereby the azido and aminogroups do not reside on the same type of base, (b) removing from theresulting double-stranded DNA the single-stranded DNA containing theazido and amino group-containing nucleotides and (c) reacting theresulting single-stranded DNA with a first modifying group which forms abond with the azido group and a second modifying group which forms abond with the amino group so as to obtain the single-stranded DNA.

This invention also provides a method for determining the nucleotidesequence of a single-stranded RNA comprising the steps of:

-   -   (a) passing the single-stranded RNA through a pore of suitable        diameter by applying an electric field to the RNA, wherein at        least each A or each G residue and at least each C or each U        residue comprises a modifying group bound to its respective base        so that each type of nucleotide in the RNA has an electronic        signature which is distinguishable from the electronic signature        of each other type of nucleotide in the RNA;    -   (b) for each nucleotide of the RNA which passes through the        pore, determining an electronic signature for such nucleotide;        and    -   (c) comparing each electronic signature determined in step (b)        with electronic signatures corresponding to each of A, G, C and        U modified as per the nucleotides in the single-stranded RNA, so        as to determine the identity of each such nucleotide,        thereby determining the nucleotide sequence of the        single-stranded RNA.

In an embodiment of the instant method, the single-stranded RNA isobtained by (a) synthesizing double-stranded RNA using a single-strandedtemplate, an RNA polymerase and nucleotides, wherein at least each A,each G, each C or each U residue comprises an azido group bound to itsbase, and at least each A, each G, each C and each U comprises an aminogroup bound to its base, whereby the azido and amino groups do notreside on the same type of base, (b) removing from the resultingdouble-stranded RNA the single-stranded RNA containing the azido andamino group-containing nucleotides and (c) reacting the resultingsingle-stranded RNA with a first modifying group which forms a bond withthe azido group and a second modifying group which forms a bond with theamino group so as to obtain the single-stranded RNA.

In another embodiment of the instant method, the single-stranded RNA isobtained by (a) synthesizing double-stranded RNA using a single-strandedtemplate, an RNA polymerase and nucleotides, wherein at least each A oreach G residue and at least each C or each U residue comprises amodifying group bound to its respective base so that each type ofnucleotide in the RNA has an electronic signature which isdistinguishable from the electronic signature of each other typenucleotide in the RNA, and (b) removing from the resultingdouble-stranded RNA the single-stranded RNA containing modifiednucleotides.

In one embodiment of the instant methods, the pore has a diameter offrom about 1 nm to about 5 nm. In a further embodiment of the instantmethods, the pore has a diameter of from about 1 nm to about 3 nm. Inembodiments of the instant methods, the pore has a diameter of about 1nm, 2 nm, 3 nm, 4 nm or 5 nm. In further embodiments, the pore is, forexample, about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.9 or 5.0 nm in diameter.

In one embodiment, a single pore is employed. In another embodiment,multiple pores are employed.

Nanopore devices are known in the art. See, for example, references [24]through [34]. Nanopores and methods employing them are disclosed in U.S.Pat. Nos. 7,005,264 B2 and 6,617,113 which are hereby incorporated byreference in their entirety.

In one embodiment of the instant methods, each A and each T or each Uresidue comprises a modifying group; each A and each U residue comprisesa modifying group; and/or each G and each C residue comprises amodifying group.

Moieties used to modify nucleotides can differ in size and/or charge, solong as each type of nucleotide in a nucleic acid whose sequence isbeing determined by the instant methods has an electronic signaturewhich differs from each other type.

DNA polymerases which can be used in the instant invention include, forexample E. Coli DNA polymerase I, Bacteriophage T4 DNA polymerase,Sequenase™, Taq DNA polymerase and 9° N polymerase (exo-)A485L/Y409V.RNA polymerases which can be used in the instant inventioninclude, for example, Bacteriophage SP6, T7 and T3 RNA polymerases.

This invention also provides a nucleotide having an azido groupcovalently bound to its base. In one embodiment, the nucleotide is dUTPand the azido group is bound to the base at the 5-position. In oneembodiment, the nucleotide is dATP and the azido group is bound to thebase at the 8-position. In another embodiment, the nucleotide is dGTPand the azido group is bound to the base at the 8-position. The azidoand amino groups can also be any other groups which permit binding of aunique moiety to each type of nucleotide.

This invention also provides a method for making a modified nucleotidecomprising contacting the instant nucleotide with an alkyne-containingcompound under conditions permitting reaction between the azido and thealkyne groups, thereby making the modified nucleotide.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

The structures of the four nucleotides are shown in FIG. 2. A and G arepurines, while C and T are pyrimidines. The overall molecular sizes of Aand G are very similar, while the sizes of C and T are similar. Thus,nanopore has been shown to be able to differentiate between purines andpyrimidines [1 and 14], but not to be able to distinguish betweenindividual purines, A and G, or between individual pyrimidines, C and T.

Disclosed here is the design of modified nucleotides to enhancediscrimination of each nucleotide by modifying A and T. Since A and Gare bulky purines similar in size, they will generate similar blockingcurrent signatures (also called electronic signatures) in the nanopore.Likewise C and T, both pyrimidines, will generate similar signatures.The site selected for modification is on the 7-position of A and the5-position of T nucleotide molecules. The 7-position of A and the5-position of T have been shown to be chemically modified with bulkygroups while not affecting basic DNA properties, such as forming thedouble-stranded DNA structure and being able to carry out polymerasereactions [2, 13 and 17]. These modifications will enlarge thediscrimination of the bases by nanopore due to the increased sizedifferences between the four nucleotides (A, G, C and T). In addition,the DNA translocation rate through the nanopore is expected to slow downdue to the bulkiness of the modified nucleotides. Thus, achieving theaccuracy and reliability required for the base-to-base sequencing isenvisioned. The overall analytical parameters in the nanoporesequencing, such as concentration of the polynucleotide, magnitude ofapplied voltage, temperature and pH value of the solution, are optimizedin order to get the most accurate and reliable results for the detectionand analysis of the DNA chain.

Use of Synthetic DNA Carrying Bulky Groups for Detection by Nanopore

In order to investigate the effect of nucleotide bulkiness on electronicblockade signals generated by the nanopore, various polynucleotides aresynthesized with different bulky groups attached to the base of thenucleotide by a DNA synthesizer. Initially, regular C's and G's are usedto synthesize a series of polynucleotides (FIGS. 3a and 3b ). Inaddition, a series of polynucleotides using modified A's(6-amino-hexylamino attached to the 8-position of the base) and modifiedT's (BIOTIN attached to the 5-position of the base) (FIGS. 3c and 3d ),which increase the bulkiness of the nucleotides, are synthesized. Theorder of the bulkiness of the nucleotides in FIG. 3 is as follows:T*>A*>G>C. These polynucleotides are then passed through the nanopore toidentify the relationship between the bulky groups attached to the baseand the difference in electronic blockade signal between the differentbases.

Attachment of Bulky Groups to Nucleotides for Nanopore Detection

(1) Design and Synthesis of Modified Nucleotides (dATP-NHCOR₁ anddUTP-NHCOR₂).

Synthesized dATP-NH₂ and dUTP-NH₂ are used as starting materials forfurther nucleotide modification while unmodified dCTP and dGTP are useddirectly (FIG. 4). The routes of nucleotide modification are shown inFIG. 5. The commercially available carboxylic acids 1˜10 will beconverted into the corresponding N-hydroxysuccinimidyl (NHS) estersconveniently using N-hydroxysuccinimide and DCC [20 and 22]. Then thenucleotides for modification (dATP-NH₂ and dUTP-NH₂) will be connectedwith the modification groups R₁ and R₂ respectively in DMF andNaHCO₃/Na₂CO₃ buffer solution [13 and 17]. After modification, the orderof nucleotide bulkiness will be: A*>U*>G>C, as purines (A and G) arelarger than pyrimidines (C and U) and in general the modification groupR₁ is larger than R₂.

(2) DNA-Extension Reaction Using Modified Nucleotides (dATP-NHCOR₁ anddUTP-NHCOR₂).

The modified dATP and dUTP, and the unmodified dCTP and dGTP, are thenbe used in a polymerase reaction to generate single-stranded DNA. Asshown in FIG. 6, after the polymerase reaction, the single-stranded DNAchain is obtained after being denatured from the template chain, whichis composed of the modified dATP and dUTP as well as unmodified dCTP anddGTP. The 5′-end of the primer chain is modified on the base by a biotinmoiety to isolate only DNA product that has incorporated the modifiednucleotides. These modified single-stranded chains are then used in thenanopore by single-channel recording techniques for sequencingsensitivity and accuracy evaluation.

DNA-Sequencing Study By Nanopore

To validate nanopore's ability to distinguish the four differentnucleotides in DNA, a series of tests are conducted as shown in FIG. 7.First, a polynucleotide stretch composed of only 50 identicalnucleotides (i) is prepared by polymerase reaction as described above.Each DNA sequence is expected to generate different electronic blockadesignatures due to the larger size difference of the nucleotides. Themodification effects of R₁ and R₂ for A and T can be compared forpreliminary optimization. Next, a polynucleotide stretch composed of 30modified A's and modified T's (ii) is prepared and then tested innanopore to demonstrate that the electronic blockade signatures differin magnitude between A and T and are easily distinguishable.

Based on the signatures generated, the candidates for R₁ and R₂ groupsare selected to achieve the best discrimination in signal. Third, ashorter polynucleotide stretch composed of 10 A's, 10 C's, 10 G's and 10T's (iii) are prepared and tested in nanopore for further confirmationon the electronic blockade signatures (also called electronicsignatures). Finally, a polynucleotide stretch composed of threeconsecutive A-C-G-T sequence (iv) is prepared and tested in nanopore.The detailed sequencing conditions can be optimized according to knownmethods. Based on these results, random DNA chain with modified A and Tand unmodified C and G is evaluated for accurate detection anddiscrimination by the nanopore. These procedures allow characterizationof the signals from each of the nucleotides and the transitions betweennucleotides of different identities. The magnitude and duration of theblockade signatures on the event diagram are then analyzed and comparedwith known diagrams for validation. The schematic of the predictedblockade signals from DNA molecules (ii), (iii) and (iv) are shown inFIG. 7. Thus, with these rational chemical designs and modifications ofthe building blocks of DNA, this invention envisions using nanopore todecipher DNA sequences at the single molecule level with single baseresolution.

Attach Small Hooks to the Nucleotides for Synthesis of DNA in PolymeraseReaction for Nanopore Detection

If a DNA polymerase is not able to synthesize a long strand of DNA dueto the bulkiness of the functional groups introduced, an alternativestrategy is to introduce small ‘hooks’ to the nucleotides, then performpolymerase reaction to produce DNA products with hook-labelednucleotides incorporated in them. The DNA products are then linked withthe large functional groups through the hook for distinct detection bynanopore.

(1) Design and Synthesis of Hook-Labeled Nucleotide dUTP-N₃.

The available dCTP, dGTP and dATP-NH₂ are used as starting materialsdirectly (FIG. 8), while dUTP-N₃ is synthesized from5-iodo-2′-deoxyuridine as shown in FIG. 9. 5-Iodo-2′-deoxyuridine isfirst coupled with propargylamine in the presence of palladium(0) andcopper(I) catalysts. Then the amino group is converted into azido groupby the diazo transfer method [11]. Finally triphosphate is introduced tothe 5′-hydroxy group of the nucleoside to yield dUTP-N₃ [6].

(2) DNA-Extension Reaction Using Hook-Labeled Nucleotides (dATP-NH₂ anddUTP-N₃).

The dATP-NH₂ and dUTP-N₃, and the unmodified dCTP and dGTP, are used inpolymerase reaction on the single-stranded nucleic acid template toobtain hook-labeled DNA products. Due to the small sizes of the azidoand amino groups, these nucleotides are expected to be good substratesof commonly used DNA polymerases. After isolation of the single strandedDNA carrying the hook, the azido groups on these modified DNA chainswill be further modified by Huisgen 1,3-dipolar cycloaddition withterminal alkynes (R₃C≡CH) in the presence of copper(I) catalyst (FIG.10) [18 and 19]. The amino groups on the “A” nucleotides of thesemodified DNA chains are connected with the modification groups R₁ in DMFand NaHCO₃/Na₂CO₃ buffer solution [13 and 17]. After modification, theorder of nucleotides bulkiness on the chain will be: A*>U*>G>C since ingeneral the modification group R₁ is larger than R₃.

Nanopore Contruction and Detection of DNA

Based on information in the art, nanopores are constructed withdifferent configurations and modifications for characterizing DNAcontaining nucleotides of different sizes.

Synthetic nanopores are described in references [24] through [28] whichare hereby incorporated by reference in their entirety. The mechanicsand kinetics of DNA passage through the pores are described inreferences [29] and [30], respectively.

Natural nanopores are described in references [31] through [34] whichare hereby incorporated by reference in their entirety.

REFERENCES

-   1. Akeson, M., Branton, D., Kasianowicz, J. J., Brandin, E. and    Deamer, D. W. Microsecond time-scale discrimination between    polycytidylic acid and polyadenylic acid segments within single RNA    molecules. Biophys. J. 1999, 77, 3227-3233.-   2. Bai, X., Kim, S., Li, Z., Turro, N. J. and Ju, J. Design and    synthesis of a photocleavable biotinylated nucleotide for DNA    analysis by mass spectrometry. Nucleic Acids Research 2004, 32(2),    535-541.-   3. Bezrukov, S. M., and Kasianowicz, J. J. Neutral polymers in the    nanopores of alamethicin and alpha-hemolysin. Biologicheskie    Membrany 2001, 18, 453-457.-   4. Chandler, E. L., Smith, A. L., Burden, L. M., Kasianowicz and    Burden, D. L. Membrane Surface Dynamics of DNA-Threaded Nanopores    Revealed by Simultaneous Single-Molecule Optical and Ensemble    Electrical Recording. Langmuir 2004, 20, 898-905.-   5. Deamer, D. W. and Branton, D. Characterization of nucleic acids    by nanopore analysis. Acc. Chem. Res. 2002, 35(10), 817-825.-   6. Lee S. E., Sidorov A., Gourlain T., Mignet N., Thorpe S. J.,    Brazier J. A., Dickman M. J., Hornby D. P., Grasby, J. A. and    Williams, D. M. Enhancing the catalytic repertoire of nucleic acids:    a systematic study of linker length and rigidity. Nucleic Acids    Research 2001, 29(7), 1565-1573.-   7. Henrickson, S. E., Misakian, M., Robertson, B. and    Kasianowicz, J. J. Driven asymmetric DNA transport in a    nanometer-scale pore. Physical Review Letters 2000, 85, 3057-3060.-   8. Kasianowicz, J. J., Brandin, E., Branton, D. and Deamer, D. W.    Characterization of individual polynucleotide molecules using a    membrane channel. Proc. Natl. Acad. Sci. USA 1996, 93, 13770-13773.-   9. Kasianowicz, J. J. Nanometer-scale pores: potential applications    for DNA characterization and analyte detection. Disease Markers    2003, 18, 185-191.-   10. Kasianowicz, J. J. Nanopore. Flossing with DNA. Nature Materials    2004, 3, 355-356.-   11. Lundquist, J. T. and Pelletier, J. C. A New Tri-Orthogonal    Strategy for Peptide Cyclization. Org. Lett. 2002, 4(19), 3219-3221.-   12. Li, L., Stein, D., McMullan, C., Branton, D.,-   Aziz, M. J. and Golovchenko, J. A. Ion-beam sculpting at nanometre    length scales. Nature 2001, 412, 166-169.-   13. Li, Z., Bai, X., Ruparel, H., Kim, S., Turro, N. J. and Ju, J. A    photocleavable fluorescent nucleotide for DNA sequencing and    analysis. Proc. Natl. Acad. Sci. USA 2003, 100, 414-419.-   14. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. and    Branton, D. Rapid nanopore discrimination between single    polynucleotide molecules. Proc. Natl. Acad. Sci. USA 2000, 97,    1079-1084.-   15. Perkins, T. T., Quake, S. R., Smith, D. E. and Chu,-   S. Relaxation of a single DNA molecule observed by optical    microscopy. Science 1994, 264, 822-826.-   16. Rief, M., Clausen-Schaumann, H. and Gaub, H. E.    Sequence-dependent mechanics of single DNA molecules. Nat. Struct.    Biol. 1999, 6, 346-349.-   17. Rosenblum, B. B., Lee, L. G., Spurgeon, S. L., Khan, S. H.,    Menchen, S. M., Heiner, C. R. and Chen, S. M. New dye-labeled    terminators for improved DNA sequencing patterns. Nucleic Acids    Research 1997, 25(22), 4500-4504.-   18. Rostovtsev, V. V., Green, L. G., Fokin, V. V. and    Sharpless, K. B. A stepwise huisgen cycloaddition process:    copper(I)-catalyzed regioselective “ligation” of azides and terminal    alkynes. Angew. Chem. Int. Ed. 2002, 41(14), 2596-2599.-   19. Seo, T. S., Bai, X., Ruparel, H., Li, Z., Turro, N. J. and    Ju, J. Photocleavable fluorescent nucleotides for DNA sequencing on    a chip constructed by site-specific coupling chemistry. Proc. Natl.    Acad. Sci. USA 2004, 101, 5488-5493.-   20. Singh, S. B. and Tomassini, J. E. Synthesis of natural flutimide    and analogous fully substituted pyrazine-2,6-diones, endonuclease    inhibitors of influenza virus. J. Org. Chem. 2001, 66(16),    5504-5516.-   21. Smith, S. B., Cui, Y. and Bustamante, C. Overstretching B-DNA:    the elastic response of individual double-stranded and    single-stranded DNA molecules. Science 1996, 271, 795-799.-   22. Streater, M., Taylor, P. D., Hider, R. C., and Porter, J. Novel    3-hydroxy-2(1H)-pyridinones. Synthesis, iron(III)-chelating    properties, and biological activity. J. Medicinal Chem. 1990, 33(6),    1749-1755.-   23. Vercoutere, W., Winters-Hilt, S., Olsen, H.,-   Deamer, D., Haussler, D. and Akeson, M. Rapid discrimination among    individual DNA hairpin molecules at single-nucleotide resolution    using an ion channel. Nat. Biotech 2001, 19, 248-252.-   24. Heng, J. B. et al., The Electromechanics of DNA in a synthetic    Nanopore. Biophysical Journal 2006, 90, 1098-1106.-   25. Fologea, D. et al., Detecting Single Stranded DNA with a Solid    State Nanopore. Nano Letters 2005 5(10), 1905-1909.-   26. Heng, J. B. et al., Stretching DNA Using the Electric Field in a    Synthetic Nanopore. Nano Letters 2005 5(10), 1883-1888.-   27. Fologea, D. et al., Slowing DNA Translocation in a Solid State    Nanopore. Nano Letters 2005 5(9),-   1734-1737.-   28. Bokhari, S. H. and Sauer, J. R., A Parallel Graph Decomposition    Algorithm for DNA Sequencing with Nanopores. Bioinformatics 2005    21(7), 889-896.-   29. Mathe, J. et al., Nanopore Unzipping of Individual Hairpin    Molecules. Biophysical Journal 2004 87, 3205-3212.-   30. Aksimentiev, A. et al., Microscopic Kinetics of DNA    Translocation through Synthetic Nanopores. Biophysical Journal 2004    87, 2086-2097.-   31. Wang, H. et al., DNA heterogeneity and Phosphorylation unveiled    by Single-Molecule Electrophoresis. PNAS 2004 101(37), 13472-13477.-   32. Sauer-Budge, A. F. et al., Unzipping Kinetics of Doubel Stranded    DNA in a Nanopore. Physical Review Letters 2003 90(23),    238101-1-238101-4.-   33. Vercoutere, W. A. et al., Discrimination Among Individual    Watson-Crick Base Pairs at the Terminin of Single DNA Hairpin    Molecules. Nucleic Acids Research 2003 31(4), 1311-1318.-   34. Meller, A. et al., Single Molecule Measurements of DNA Transport    Through a Nanopore. Electrophoresis 2002 23, 2583-2591.

What is claimed is: 1-17. (canceled)
 18. A single stranded DNAcomprising one or more modified nucleotide analog, wherein, when anelectric field is applied to the DNA, it produces a unique electronicsignature when said DNA interacts and passes through a pore of suitablediameter.
 19. The single stranded DNA of claim 1, wherein, when anelectric field is applied to the DNA, each modified nucleotide analogproduces an electronic signature which is distinguishable from that ofeach other type of nucleotide or nucleotide analog in the DNA when saidmodified nucleotide analog interacts and passes through a pore ofsuitable diameter.
 20. The single stranded DNA of claim 1, wherein theone or more modified nucleotide analog comprises a modification locatedon the base of the nucleotide analog.
 21. The single stranded DNA ofclaim 2, wherein the one or more modified nucleotide analog comprises amodification located on the base of the nucleotide analog.